(Supported in part by NIH AI 36982-01 to K. McDonough) We are studying the intracellular localization of tubercle bacilli (Mtb) within macrophages. We previously reported (McDonough, et al. (1993) Infec. Immun. 612763) that virulent tubercle bacilli escaped from phagolysosomes into nonfused vesicles or the cytoplasm at several days post-infection. Our recent studies show that these "extraphagosomal" bacteria appear sooner (by 4th day post infection) and in higher numbers when virulent Mtb bacteria are pre-grown within macrophages. These findings are significant because the immune response required to control bacteria with unrestricted access to the host cell's cytoplasm is vastly different from that needed to eradicate a bacterium sequestered within a phagolysosome. It has been difficult to unambiguously differentiate, using traditional TEM techniques, between bacteria that are truly free in the cytoplasm and those that may be encased within a very tightly associated vacuolar membrane. Our approach has been criticized (see Xu, et al. (1994) J. Immunol. 153:2568) by others using cryo electron microscopy. The increased sample size and depth of field provided by HVEM, as well as the ability to perform 3-D image reconstructions should help us to better define the nature of the membranes (if any) that surround these tubercle bacilli. We have been comparing HVEM stereo pairs of macrophage lysates containing virulent tubercle bacteria with those containing avirulent tubuercle bacteria to determine the nature of the vacuoles surrounding these bacteria. A virulent bacillis calmette guerin (BGC) bacteria are often found enclosed within well-preserved contiguous membrane sacs, whereas virulent M. tuberculosis bacteria are more often found within disrupted membranes. These differences are currently being quantified and analyzed. Reconstructions from Sterecon tracings of 0.75lm thick sections photographed in the HVEM were made for poster presentation. Two posters were presented: K.A. McDonough, M. Florczyk, Y. Kress, J. Turner and B.R. Bloom 1997 Passage within macrophages affects the intracellular localization of virulent tubercle bacilli upon reinfection, Proc. ASM Conference on Tuberculosis. M. A. Florczyk, James N. Turner, Karolyn F. Buttle and Kathleen A. McDonough 1997 High Voltage Electron Microscopic Analysis of the Intracellular Localization of Mycobacterium Tuberculosis within Macrophages, Porc. Microbial Infectious Disease in Host Response, Cold Spring Harbor.